This invention relates to a monoclonal antibody which recognizes specifically an O-acylsialic acid containing sugar chain, particularly 4-O-acetyl-N-glycolylneuraminic lactosylceramide (hereinafter called: "GM.sub.3 (4-O-Ac-NeuGc)" and a method for preparing a hybridoma capable of producing said antibody.
Glycolipid is a component of cell membrane, and a diversity of molecular species exist according to the differences of the kinds, the number and the binding manner of the constituting sugars, exhibiting species specific, organ specific and cell specific distributions. It is clarified that the glycolipid functions to play an important role as concerned with control of growth or differentiation or cellular interaction, in addition to such actions as receptors for bacterial toxins, hormones, etc. and also immunological determinants such as blood group substances, etc.. Further, it is shown that with accompaniment of transformation of cells, qualitative and quantitative changes in its composition occur, whereby a part of the glycolipids can become cancer antigens, and also it is suggested that its compositional change may participate directly in the canceration mechanism, such as examples in which some glycolipids function as regulators for cell growth mechanism through growth factors and protein kinase.
On the other hand, a method for establishing cell strain producing uniform antibody having specificity for one kind of antigenic determinant was reported by Milstein et al [Nature 256, 495 (1975)], whereby it has become possible to determine qualitatively and quantitatively a minute amount of substance. For performing screening of cancer antigents, many monoclonal antibodies specific for cancer cells were prepared by use of this technique, and among them, some were clarified to be antibodies which recognize sugar chains of glycolipids or glycoproteins [J. Natl, Cancer Inst., 71, 231 (1983)].
For example, antibodies reactive with glycolipids such as GD.sub.2 ganglioside or GD.sub.3 ganglioside have been obtained as the monoclonal antibody to human melanoma. The monoclonal antibody NS19-9 which is specific for pancreatic cancer is reactive with glycolipids and glycoproteins having sialosyl Lewis A type sugar chain. These antibodies are useful for diagnosis and observation of therapeutical course, and also further attempted to be utilized for therapy. The qualitative and quantitative changes of glycolipids accompanied with canceration are caused by changes in activities of various glycosyl-transferases in the sugar chain biosynthesis mechanism by abnormal expression of gene, with the result that sugar chain structures which do not exist in normal tissues are produced. Such sugar chain structures can be utilized as cancer marker.
Thus, as a clue to clarification of canceration mechanism as well as a cancer antigen and a cancer marker, importance and usefulness of glycolipid are attracting attention, and its application to the clinical field such as diagnosis, therapy, etc. is expected.
Of the glycolipids, those containing, in the sugar chain, sialic acid which is an acidic sugar are comprehensively called gangliosides. Sialic acids may be broadly classified into N-acetylneuraminic acids and N-glycolylneuraminic acids, and further those having the hydroxyl groups of the respective sialic acids acylated also exist.
In recent years, it has been clarified that a complex sugar containing 9-O-acetylated N-acetylneuraminic acid functions as the receptor for Influenza C type [J. Biol. Chem., 261, 5947 (1986)]. Also, as the human cancer antigen which is recognized by the monoclonal antibody specifically reactive with human melanoma cells, GD.sub.3 ganglioside O-acetylated at the 9-position of sialic acid was identified [J. Biol. Chem., 259, 7453 (1984)]. Thus, O-acylsialic acid containing chain and the antibody thereto are expected to be applied to diagnosis and therepy of various diseases including cancer.
On the other hand, while sialic acids are broadly detected in various organs, cells, body fluids of animal species, N-glycolylneuraminic acid has not yet been found in normal human being and chicken.
Heterophile antibody which is found in serum sickness patients and aggregates red blood cells of sheep, horse, pig, rabbit, guinea pig is called Hanganatziu-Deicher (hereinafer called "H-D" antibody. An antigen recognized by this antibody is called H-D antigen N-glycolylneuraminicacd containing ganglioside was reported to have H-D antigen activity [Biochem. Biophys. Res. Commun., 79, 388 (1977)], and the sugar chain structure of NeuGc .alpha.2-3 Gal was identified as the main antigenic determinant.
In recent years, an antibody reactive with various gangliosides having H-D antigen activity was prepared from the serum of a chicken immunized with N-glycolylneuraminyl lactosylceramide (hereinafter called GM.sub.3 (NeuGc)) which is a ganglioside having H-D antigen activity [Molec. Immun., 19, 87 (1982)]. Further, by use of this antibody, N-glycolylneuraminic acid was reported to exist characteristically in human cancer tissue [Biken J., 25, 47 (1982)]. Also, in the glycolipid extracted from human colon cancer tissue, several kinds of H-D antigen active N-glycolylneuraminic acid containing gangliosides were detected, and from among the tissues of teratoma, a glycoprotein having H-D antigen active sugar chain was detected [Gann, 75, 1025 (1984)]. When the glycolipid having H-D antigen activity detected from human colon cancer tissue by use of the antibody prepared in chicken was analyzed, GM.sub.3 having O-acylated glycolyl-neuraminic acid was detected, in addition to GM.sub.2 (NeuGc), GM.sub.3 (NeuGc), IV.sup.3 NeuGc-nLcOse4 Cer. From the reactivity of the antibody prepared in chicken, the ganglioside was estimated to be GM.sub.3 (4-O-Ac-NeuGc), which is glycolyl-neuraminic acid acetylated at the 4-position. For confirmation of this fact, a polyclonal antibody which reacts with GM.sub.3 (1-O-Ac-NeuGc) but does not crossreact with GM.sub.3 (NeuGc) was prepared in chicken, and human colon cancer tissue was investigated by use of this, whereby GM.sub.3 (4-O-Ac-NeuGc) was surely found [Molec. Immun., 23, 631 (1986)].
Thus, some kinds of O-acylsialic acid containing sugar chains, particularly GM.sub.3 (4-O-Ac-NeuGc), are considered to be cancer-associated antigens, and detection thereof with high sensitivity and good precision is extremely important in cancer diagnosis. For detecting such sugar chain antigen with good efficiency, immunological assay methods are considered to be excellent in aspects of detection sensitivity and precision.
Although there is substantially no report about a polyclonal antibody to O-acylsialic acid containing sugar chain, the antibody reactive with GM.sub.3 (4-O-Ac-NeuGc) was obtained in the prior art by immunizing chicken with a purified glycolipid antigen and separating from its serum [Molec. Immun., 23, 631 (1986)]. However, this method has some drawbacks. That is, (1) for obtaining antiserum, a large amount of purified antigens is required every time; (2) there are variances in affinity and activity caused primarily by individual differences between the immunized animals; (3) cumbersome operations are necessary for purification of the desired antibody, since antibodies other than the desired antibody also exist as mixed therewith; (4) the amount prepared at once is limited, etc. Therefore, for performing immunological assay correctly and with the maximum effect, it has been desired to supply a large amount of uniform antibodies of stable quality without mixing of other antibodies. Preparation of such antibodies has been already reported as the monoclonal antibody producing technique.
However, concerning monoclonal antibodies reactive specifically with O-acylsialic acid containing sugar chains and hybridomas having ability to produce said monoclonal antibodies are reported only on GD.sub.3 acetylated at the 9-position of N-acetylneuraminic acid as mentioned above, and there has been no report on a derivative having hydroxyl group other than at the 9-position substituted or hydroxyl group of N-glycolylneuraminic acid substituted.